Connectivity mapping is a well-known hypothesis generating and testing tool having successful application in the fields of operations research, computer networking and telecommunications. The undertaking and completion of the Human Genome Project, and the parallel development of very high throughput high-density DNA microarray technologies enabling rapid and simultaneous quantization of cellular mRNA expression levels, resulted in the generation of an enormous genetic database. At the same time, the search for new pharmaceutical actives via in silico methods such as molecular modeling and docking studies stimulated the generation of vast libraries of potential small molecule actives. The amount of information linking disease to genetic profile, genetic profile to drugs, and disease to drugs grew exponentially, and application of connectivity mapping as a hypothesis testing tool in the medicinal sciences ripened.
The general notion that functionality could be accurately determined for previously uncharacterized genes, and that potential targets of drug agents could be identified by mapping connections in a data base of gene expression profiles for drug-treated cells, was spearheaded in 2000 with publication of a seminal paper by T. R. Hughes et al. [“Functional discovery via a compendium of expression profiles” Cell 102, 109-126 (2000)], followed shortly thereafter with the launch of The Connectivity Map (-map Project by Justin Lamb and researchers at MIT (“Connectivity Map: Gene Expression Signatures to Connect Small Molecules, Genes, and Disease”, Science, Vol 313, 2006.) In 2006, Lamb's group began publishing a detailed synopsis of the mechanics of C-map construction and installments of the reference collection of gene expression profiles used to create the first generation C-map and the initiation of an ongoing large scale community C-map project, which is available under the “supporting materials” hyperlink at http://www.sciencemag.org/content/313/5795/1929/suppl/DC1.
The basic paradigm of predicting novel relationships between disease, disease phenotype, and drugs employed to modify the disease phenotype, by comparison to known relationships has been practiced for centuries as an intuitive science by medical clinicians. Modern connectivity mapping, with its rigorous mathematical underpinnings and aided by modern computational power, has resulted in confirmed medical successes with identification of new agents for the treatment of various diseases including cancer. Nonetheless, certain limiting presumptions challenge application of C-map with respect to diseases of polygenic origin or syndromic conditions characterized by diverse and often apparently unrelated cellular phenotypic manifestations. According to Lamb, the challenge to constructing a useful C-map is in the selection of input reference data which permit generation of clinically salient and useful output upon query. For the drug-related C-map of Lamb, strong associations comprise the reference associations, and strong associations are the desired output identified as hits.
Noting the benefit of high-throughput, high density profiling platforms which permit automated amplification, labeling hybridization and scanning of 96 samples in parallel a day, Lamb nonetheless cautioned: “[e]ven this much firepower is insufficient to enable the analysis of every one of the estimated 200 different cell types exposed to every known perturbagen at every possible concentration for every possible duration . . . compromises are therefore required” (page 54, column 3, last paragraph). Hence, Lamb confined his C-map to data from a very small number of established cell lines. This leads to heightened potential for in vitro to in vivo mismatch, and limits information to the context of a particular cell line. Selection of cell line, therefore, may be critical to the utility of a resulting C-map.
Lamb stresses that particular difficulty is encountered if reference connections are extremely sensitive and at the same time difficult to detect (weak), and Lamb adopted compromises aimed at minimizing numerous, diffuse associations. Since the regulatory scheme for drug products requires high degrees of specificity between a purported drug agent and disease state, and modulation of disease by impacting a single protein with a minimum of tangential associations is desired in development of pharmaceutical actives, the Lamb C-map is well-suited for screening for potential pharmaceutical agents despite the Lamb compromises.
The connectivity mapping protocols of Lamb would not be predicted, therefore, to have utility for hypothesis testing/generating in the field of cosmetics. Cosmetic formulators seek agents or compositions of agents capable of modulating multiple targets and having effects across complex phenotypes and conditions. Further, the phenotypic impact of a cosmetic agent must be relatively low by definition, so that the agent avoids being subject to the regulatory scheme for pharmaceutical actives. Nonetheless, the impact must be perceptible to the consumer and preferably empirically confirmable by scientific methods. Gene transcription/expression profiles for cosmetic conditions are generally diffuse, comprising many genes with low to moderate fold differentials. Cosmetic agents, therefore, provide more diverse and less acute effects on cellular phenotype and generate the sort of associations expressly taught by Lamb as unsuitable for generating connectivity maps useful for confident hypothesis testing.
Contrary to the teachings of Lamb and the prior art in general, the present inventors surprisingly discovered that useful connectivity maps could be developed from cosmetic active—cellular phenotype—gene expression data associations in particular with respect to skin care cosmetics. Specifically, certain aspects of the present invention are based on the surprising discovery that selection of human dermal fibroblast cells as the relevant cell line resulted in construction of connectivity maps useful for hypothesis generating and testing relating to cosmetic agents in treatment of photo-damaged/photo-aged skin, while a combination of fibroblast and keratinocyte cells appeared most suitable for intrinsically aged skin. Skin is a highly complex system, and the effects of aging conditions, whether intrinsic or photo-induced, on skin are diffuse and not fully understood. Therefore, it could not be predicted that a fibroblast cell or a keratinocyte cell, or any combination thereof, could be used to construct a connectivity map effective for generating and testing hypotheses relating to cosmetic actives and genes associated with skin aging.
Skin is a complex, multi-layered and dynamic system that provides a protective covering defining the interactive boundary between an organism and the environment. It is the largest organ of the body and is vitally important to both our health and our self image. As shown in FIG. 1, skin comprises three principal layers, the epidermis, the dermis, and a layer of subcutaneous fat. The majority of cells in the epidermis are keratinocytes that produce a family of proteins called keratins. Keratins contribute to the strength of the epidermis. The epidermis itself may be divided into multiple layers with the outermost layer referred to as the stratum corneum, and the innermost layer referred to as the basal layer. All epidermal cells originate from the basal layer and undergo a process known as differentiation as they gradually displace outward to the stratum corneum, where they fuse into squamous sheets and are eventually shed. In healthy, normal skin, the rate of production equals the rate of shedding (desquamation).
The differentiating epidermal cells form distinct though naturally blended layers. As the cells displace outward, they flatten and become joined by spiny processes forming the stratum spinosum, or spinous layer. The cells manufacture specialized fats called sphingolipids, and begin to express keratins associated with terminal differentiation. As keratin is produced, it is incorporated into the cellular matrix, strengthening the skin and providing structural support to the outer layers. As the cells migrate further outward and develop characteristic granules that contain proteins which contribute to the aggregation of keratins; they now form part of the granular layer. Cells lose their nuclei in the outer part of this layer, and the granules release their contents contributing to cornification. Vesicles containing lipids discharge into the spaces between the cells, creating a barrier structure that has been suggested to function like bricks (cells) and mortar (lipids). As the cells rise into the outermost layer of the epidermis—the stratum corneum, sometimes called the horny layer or the cornified layer—they take the form of flattened discs, tightly packed together. These flattened cells, called corneocytes, are effectively dead. The lipids of the epidermis play an important role in maintaining skin health, as they help the stratum corneum to regulate water loss while providing a virtually impermeable hydrophobic barrier to the environment. Fully mature keratinocytes function to protect the skin from UV light damage, and help effectuate immune response to environmental stimuli.
The dermis, which lies just beneath the epidermis, is composed largely of the protein collagen. Most of the collagen is organized in bundles which run horizontally through the dermis and which are buried in a jelly-like material called the ground substance. Collagen accounts for up to 75% of the weight of the dermis, and is responsible for the resilience and elasticity of skin. The collagen bundles are held together by elastic fibers running through the dermis. The fibers are comprised of a protein called elastin, and make up less than 5% of the weight of the dermis. Fibroblasts function to synthesize collagen and the dermis ground substance, including components glycoproteins and glycosaminoglycans such as hyaluronic acid (which is able to bind water). The junction between the epidermis and the dermis is not straight but undulates—more markedly so in some areas of the body than others. A series of finger-like structures called rete pegs project up from the dermis, and similar structures project down from the epidermis. These projections increase the area of contact between the layers of skin, and help to prevent the epidermis from being sheared off. As skin ages, the projections get smaller and flatter. Networks of tiny blood vessels run through the rete pegs, bringing nutrients, vitamins and oxygen to the epidermis, although the epidermis itself is avascular and nourished by diffusion from the rete pegs. The dermis also contains the pilobaceous units comprising hair follicles and sebaceous glands, apocrine and eccrine sweat glands, lymphatic vessels, nerves, and various sensory structures, including the mechano-sensing Pacinian and Meissner's corpuscles.
Beneath the dermis lies the hypodermis, which comprises subcutaneous fat that cushions the dermis from underlying tissues such as muscle and bones. The fat is contained in adipose cells embedded in a connective tissue matrix. This layer may also house the hair follicles when they are in the growing phase.
Thus, skin is a multilayered complex organ comprising a wide variety of cellular types and structures, including epidermal and dermal connective tissue with blood and lymphatic vessels, the pilosebaceous units, glands, nerves, various sensory structures, the hypodermal adipose tissue, and the elastic fascia beneath the hypodermis. In turn, these structures are composed of a number of different cell types including keratinocytes, melanocytes, neuroendocrine Merkel cells, sebocytes, fibroblasts, endothelial cells, pericytes, neurons, adipocytes, myocytes and resident immunocytes including Langerhans cells, other dendritic cells, T cells and mast cells. Two of the main cell lineages in the skin are epithelial cells, which in general form the linings of the body and the parenchyma of many organs and glands, and mesenchymal cells, which form connective tissue, blood vessels and muscle. Dermal fibroblasts are mesenchymal cells, and keratinocytes are epithelial cells, which comprise most of the structure of the epidermis.
Skin aging is likewise a complex multi-factorial process that results from unrepaired cellular and tissue damage leading to impaired functional capacity. The aging process in skin is the result of both intrinsic and extrinsic factors occurring over decades. Skin is subject to many of the same intrinsic aging processes as other organs, but is also exposed to solar radiation, an important extrinsic factor that contributes to premature skin aging or photo-aging. Another important extrinsic factor potentially contributing to skin aging is smoking. There have been major advances in the understanding of the aging process with the identification of cellular pathways and genes associated with longevity and aging. Several theories have been proposed to explain intrinsic aging, including cellular senescence resulting from telomere shortening, mutations in nuclear and mitochondrial DNA, hormonal insufficiency and oxidative stress. Reactive active oxygen species and the direct effects of ultraviolet radiation (UVR) appear to play major roles in photo-aging. As is the case for aging in general, an integrated understanding of skin aging has not been developed.
Skin researchers have categorized age-inducing factors as either intrinsic or extrinsic, although these are interdependent, reflected for example by the fact that extrinsic factors may accelerate intrinsic aging. One example of the complex interplay of factors involves free radicals, which are both generated internally through normal metabolic processes and produced as a consequence of external factors, including UVR exposure. As a result of the age-associated decline in protective internal antioxidant mechanisms, free radicals can reach higher and sustained levels in cells and alter both proteins and DNA in skin. Levels of altered protein and DNA may accumulate causing damage. In addition, ongoing accumulation of damage secondary to internally-generated free radicals combined with those generated from UVR and other external assaults (surfactants, allergens, and other irritants) can promote a chronic inflammatory state. This chronic inflammation compromises skin health and may accelerate the aging process; for example, proteolytic enzymes are produced, resulting in collagen degradation. Activated inflammatory cells resulting from elevations in circulating pro-inflammatory mediators (e.g., prostaglandins, cytokines, histamines) produce reactive oxygen species that can cause oxidative damage to nucleic acids, cellular proteins and lipids. Accumulated damage caused by reactive oxygen species may stimulate a host of cytokine cascades that results in photo-aging and photo-carcinogenesis. These changes may be tied to the appearance of aging skin.
Other changes resulting from the complex interplay between intrinsic and extrinsic factors that may impact the appearance of fine lines, wrinkles and texture include the following:                Epidermal thickness and cellular turnover rate of both the epidermis and the stratum corneum declines and epidermal differentiation is altered.        The dermis becomes thinner as major structural molecules including collagen, elastin and glycosaminoglycans decrease in amount. The elastic network in photo-damaged skin becomes disorganized and aggregated and the various structural proteins may be modified by glycation. Metalloproteinase activity increases in photo-damaged skin, contributing to the degradation of collagen and elastin.        Convolution of dermal-epidermal junction (rete ridges) flattens with age, resulting in a loss of mechanical strength. This also leads to decreased microcirculation to the upper dermis and, thus, decreased nourishment to the epidermis.        Age-related changes in inter- and intra-cellular signaling lead to decreases in collagen synthesis.        Changes in hyaluronic acid content within the skin occur with age. Hyaluronic acid is a natural moisturizer within the skin, binding up to 1000 times its weight in water. Age-related declines result in compromised moisturization and firmness.        Decreased intracellular energy sources including ATP and NADH lead to an inability of skin cells to sustain youthful skin biochemistry, thereby reducing the skin's ability to maintain and restore youthful skin structure.        In the epidermis, a lack of estrogen slows the activity of the basal keratinocytes, and consequently leads to epidermal atrophy. This atrophic, fragile skin is less well protected by the normal surface film of lipids, because of the slow decline in sebum secretion experienced by everyone as they age. The stratum corneum barrier is less effective, and the skin may develop reactions to irritants, particularly if skin care has been inadequate or too aggressive.        The time necessary to repair the stratum corneum barrier increases considerably with age: the replacement of skin cells takes about twice as long for people over 75 as for those around 30.        
These changes may compromise skin's elasticity, firmness and structure—contributing to areas of collapse and irregularity and ultimately manifesting as fine lines, wrinkles and texture problems.
There are many skin care products available to consumers which are directed to improving the health and/or physical appearance of skin tissue, such as keratinous tissue. Many of such products are directed to delaying, minimizing or even eliminating changes typically associated with one or both of aging and environmental damage to skin. Such products may include one or more of the numerous cosmetic agents known to be useful in improving the health and/or appearance of keratinous tissue. Although many such agents are known, an ongoing need exists to identify cosmetic agents that can provide new or improved benefits to skin tissue. There is also a need to identify additional cosmetic agents that provide similar or improved benefits as compared to existing products but which are easier to formulate, produce, and/or market.
Successful identification of anti-aging cosmetic agents has proven to be difficult due to the multi-cellular, multi-factorial processes that occur in skin over the course of decades. In addition, many desirable cosmetic agents may comprise a mixture of compounds with effects and interactions that may not be fully understood. This is often the case with a botanical or other natural extract that may affect many cellular/pathways. An additional challenge for cosmetic formulators is that cosmetics must be very safe and adverse effects generally are not acceptable. Further, while much is known about skin aging, there is much that is still poorly understood or unknown. Conventional in vitro studies of biological responses to potential cosmetic agents involve testing hundreds or thousands of potential agents in various cell types before an agent that gives the desired result can be identified and moved into a next stage of testing. However, such studies can be hindered by the complex or weakly detectable responses typically induced and/or caused by cosmetic agents. Such weak responses arise, in part, due to the great number of genes and gene products involved, and cosmetic agents may affect multiple genes in multiple ways. Moreover, the degree of bioactivity of cosmetic agents may differ for each gene and be difficult to quantify.
For example, niacinamide is a well-known cosmetic agent producing skin benefits such as improved barrier function and anti-inflammatory activity. Niacinamide is a precursor of NADH, which is involved in more than 100 reactions in cellular metabolism. In contrast to drug agents, which are selected for specificity and which are intended to have measurable effects on structure and function of the body, cosmetic agents are selected for effect on appearance and may not effect structure and function of the body to a measurable degree. Cosmetic agents tend to be non-specific with respect to effect on cellular phenotype, and administration to the body is generally limited to application on or close to the body surface.
The value of a connectivity map approach to discover functional connections among cosmetic phenotypes such as aged skin, gene expression perturbation, and cosmetic agent action is counter-indicated by the progenors of the drug-based C-map. The relevant phenotypes are very complex, the genetic perturbations are numerous and weak, and cosmetic agent action is likewise diffuse and by definition, relatively weak. It is unclear whether statistically valid data may be generated from cosmetic C-maps and it is further unclear whether a cell line exists which may provide salient or detectable cosmetic data.
Surprisingly, the present inventors have provided a C-map approach that is generalizable and biologically relevant for identification of potential cosmetic actives, and demonstrate that the C-map concept is viable by use of benchmark cosmetic actives to query the reference data and by identification of new cosmetic actives.